|Budget Amount *help
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1990: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1989: ¥5,000,000 (Direct Cost: ¥5,000,000)
In order to prepare a large amount of recombinant human TPO (rhTPO), we constructed an expression plasmid, pSV2-HTPO. dhfr, containing both human thyroid peroxidase and mouse dihydrofolate reductase cDNAs and transfected into chinese hamster ovary cells. A subline producing a large amount of rhTPO was established by selecting clones resistant to methotrexate. The molecular weight of the rhTPO was the same as purified hTPO (native TPO). This expressed protein had peroxidase activity when determined by guaiacol oxidation.
When we compared immunoreactivity of rhTPO with that of native TPO using competitive binding inhibition and dilution tests in micro-ELISA system, rhTPO had almost the same immunoreactivity as native hTPO. In respect to background absorbance, rhTPO was superior to native hTPO. Since international standard serum for anti-TPO autoantibodies was introduced into this micro-ELISA system using rhTPO as antigen to measure anti-TPO autoantibodies, we have been able to express ant
i-TPO autoantibody concentration as IU/ml. Using about one hundred sera from patients with thyroid-related disorders, we examined the correlation between titer of microsome test and anti-TPO antibody concentration. In the sera without anti-thyroglobulin in autoantibodies (less tha X100 in thyroid test), a significant correlation was observed between them, although no correlation was calculated using all sera. The above result was consistent with our theme that a kit measuring autoantibodies specific for TPO should be developed.
To study His residue binding to heme iron, we expressed both hTPO・cDNAs, longer cDNA consisting of 3,048 nucleotides and shorter cDNA lacking the 10th exon. The former cDNA showed both hTPO protein and TPO activity, when it was introduced to vaccinia virus expression system. The latter cDNA, however, could not express TPO activity. From the above results, we considered two His residues contained in the 8th and 10th exons as proximal and distal His residues, respectively, among three His residues belonging to three respective exons, the 8th, 9th, and 10th. However, a possibility that conformationl change in shorter cDNA expression might disturb TPO activity expression remains to be solved. Less